The mechanism of mRNA degradation and stabilization is being examined in murine T cells. The model system chosen for study is the regulation of the GM-CSF gene by phorbol esters which has been shown to occur predominantly by stabilization of the mature transcript in the cytoplasm. Portions of the GM-CSF gene coding and 3' untranslated regions have been substituted for the SV40 poly A addition signals in RSVCAT and the resulting hybrid constructs studied in transient assays in a variety of cell lines. CAT assays and mRNA analyses have been performed. These studies show that GM-CS mRNA degradation is mediated in part by the AU sequences in the 3'UTR but that other sequences within the mRNA also contribute importantly to mRNA instability. Further, mRNA degradation mediated by all of these sequences is not cell-type or species specific. Transient assays in murine T cells show that TPA-mediated stabilization of GM-CSF mRNA is not mediated by the AU boxes but that other portions of the mRNA must respond to TPA. TPA stabilization in transient assays is restricted to T cells and does not occur, for example, in fibroblasts. RNA band shift assays have been used to identify cytoplasmic proteins that are responsible for mediating mRNA degradation and stabilization. Work over the next year will concentrate on generating and studying the effects of linker scanner mutations within the GM-CSF mRNA in order to map precisely the sequences responsible for the above observations.